The template dependent PCR (Polymerase Chain Reaction) method for synthesizing nucleic acid has been a great driving force for study in the recent bioscience field. The PCR method enables the exponential amplification a nucleic acid comprising a nucleotide sequence complementary to a template nucleic acid using a small amount the template. The PCR method prevails widely at present as a tool for cloning or detecting a gene. In the PCR method, a pair of primers, comprising a complementary nucleotide sequence, is used for both ends of the target nucleotide sequence. The primer pair is designed such that one primer anneals to an extension product provided by another primer. A synthesis reaction proceeds by repeating an annealing to the mutual extension product and a complementary strand synthesis reaction, and an exponential amplification is thus attained.
In the PCR method, a single-stranded nucleic acid template is made by some method and a primer is annealed to the template. Since a template dependent DNA polymerase requires a primer as a replication origin, the preparation of the single-stranded template is considered to be essential, in order to anneal the primer to it in the PCR method. The step of converting a double-stranded template nucleic acid to a single-strand is generally called denaturing. The denaturing is usually carried out by heating. Since other reaction components required for the synthesis of nucleic acid, including DNA polymerase, are heat resistant, the denaturing and successive complementary strand synthesis reactions can be carried out by combining all of the reaction components and further heating the reaction mixture. However, the conventional methods containing the heat treatment step have the problems described below.
First, in the PCR method, the denaturing of double-stranded nucleic acid and the annealing of a primer must be performed in each cycle. For that purpose, a specific mechanism for controlling temperature is required. For example, although a method for monitoring the increase of a reaction product during PCR has been developed, the method cannot be carried out using conventional analytical equipment and, therefore, it is necessary to provide dedicated equipment having a mechanism for controlling temperature to carry out the PCR method as well as a mechanism for monitoring the reaction. Accordingly, if all reactions for the nucleic acid synthesis could be carried out at a constant temperature, the reaction could be monitored easily using conventional analytical equipment. Such a convenient method would simplify not only the equipment but also experimental operation. However, a reaction principle for this method is not currently known.
The reaction specificity of PCR depends on the specificity of primer annealing. A primer can be expected to anneal to a single-stranded nucleic acid with adequate specificity at a high temperature, near a melting temperature. When the temperature is not adequately high, non-specific annealing, and resulting non-specific complementary strand synthesis reactions, often occur. Since the PCR method is accompanied by a complicated temperature change, the reaction mixture may possibly be exposed to a temperature at which a non-specific reaction is apt to occur. This is one of the causes for the non-specific reactions associated with the PCR method.
Several methods for solving the problem of the temperature-dependent non-specific reaction have been proposed. For example, one practically used method uses a DNA polymerase that does not work at a certain temperature or less. Specifically, a temperature sensitive DNA polymerase inhibitor, an antibody against the DNA polymerase, or a variant of DNA polymerase and the like are reportedly used. Further, a method in which the reaction components are put in compartments separated by a partition that is meltable at a high temperature, so that the components are mixed only after heated to an adequate temperature, is also known. At all events, since the PCR method accompanies a complicated temperature change, it is required to use a special component for preventing the non-specific reaction.
Methods of amplifying DNA having a complementary sequence to a target sequence using the target sequence as a template, such as the Strand Displacement Amplification (SDA) method, are also known (Pro.N.A.S., 89, pp. 392-396; 1992, Nucleic Acid, Res., 20, pp. 1691-1696; 1992). In the SDA method, when a complementary strand is synthesized using as a synthesis origin a complementary primer to the 3′-side of a certain nucleotide sequence, a unique DNA polymerase enables synthesis of a complementary strand that displaces the double-strand region at the 5′-side. When reciting “5′-side” or “3′-side” hereinafter, the terms mean the direction of a template strand. This method is called Strand Displacement Amplification because the double-strand portion of the 5′-side is displaced with a complementary strand which has been newly synthesized.
In the SDA method, the step of changing temperature, which is essential for the PCR method, can be omitted by inserting a restriction enzyme recognition sequence in a sequence to which a primer anneals. Namely, a nick provided by the restriction enzyme gives a 3′-OH group that becomes the origin of complementary strand synthesis. The strand displacement and complementary strand synthesis are carried out from the origin and the complementary strand synthesized is dissociated as a single-strand and utilized as the template in the subsequent complementary strand synthesis. Thus, the SDA method does not require a complicated temperature control that has been essential for the PCR method.
Although the temperature control is not necessary in the SDA method, the heat treatment is still necessary to prepare the single-strand needed for primer annealing when double-stranded nucleic acid is used as a template. Further, this method requires both a restriction enzyme that provides a nick and a DNA polymerase with strand displacement activity. Necessity of an additional enzyme leads to an increase in cost. Furthermore, in order to introduce a nick and to not cleave the double-strand (i.e., only one strand is cleaved), a dNTP derivative, such as α-thio dNTP, must be used as a substrate for synthesis so that one of the double-strands has resistance to enzyme digestion. Accordingly, an amplified product obtained by SDA has a configuration different from natural nucleic acid. Thus, restriction enzyme cleavage and use of an amplified product in gene cloning are limited. The use of the dNTP derivative also causes an increase in cost.
As a method for amplifying nucleic acid without a complicated temperature control, Nucleic Acid Sequence-based Amplification (NASBA), which is also called TMA/Transcription Mediated Amplification method, is known. NASBA is a reaction system in which DNA synthesis is carried out using DNA polymerase, a target RNA as a template, and a probe to which T7 promoter has been added. The synthesized DNA is made double-stranded using a second probe, and transcription is performed using T7 RNA polymerase. The double-stranded DNA obtained is used as a template, thereby amplifying a large quantity of RNA (Nature, 350, pp. 91-92, 1991). Transcription using T7 RNA polymerase in NASBA proceeds isothermally. However, NASBA requires RNA as a template, and thus cannot be applied to double-stranded nucleic acid. If the double-stranded nucleic acid is made single-stranded, this reaction can be performed; however, in this case, a complicated temperature control similar to PCR is needed. Further, a combination use of plural enzymes, such as reverse transcription enzyme, RNaseH, DNA polymerase, and T7 RNA polymerase, is essential, which is economically disadvantageous as in SDA. Thus, known nucleic acid amplification reaction methods have the problem of complicated temperature control or the necessity to use plural enzymes.
In order to solve the problem of the temperature control in known nucleic acid synthesis methods, a complementary strand has been synthesized under a specific condition, using a primer as the origin for the synthesis (Published Japanese Translation of International Publication No. Hei 11-509406; WO97/00330). The method recognizes of the fact that the hybridization of nucleic acids having complementary nucleotide sequences occurs in a state of dynamic equilibrium (kinetics). In this prior art method, it is believed that the complementary strand synthesis reaction, using a primer as the origin for the synthesis, may occur at a certain probability, even at a temperature that causes complete denaturing or below. The term “complete denaturing” as used herein means a condition in which most of the double-stranded template nucleic acid becomes single-stranded.
In this report, when a primer and a DNA polymerase with strand displacement activity are combined with the double-stranded template nucleic acid and the temperature is raised, the synthesis of complementary strand was observed at a temperature which did not cause denaturation of a template nucleic acid. However, the reaction efficiency of the complementary strand synthesis without thermal cycling is remarkably lower than that obtained in the PCR method with thermal cycling. In fact, the present inventors performed a supplementary test and confirmed that the reaction had certainly occurred, but the amount of the reaction product obtained by this method did not reach a usable level of a practical nucleic acid synthesis method.
As described above, a nucleic acid synthesis reaction without controlling the temperature and deteriorating the specificity and efficiency of the reaction, has not yet been reported.